KR101890213B1 - Carbonate polymer with disulfur five-membered ring functional group on side chain and application thereof - Google Patents

Abstract

The present invention discloses a biodegradable polymer containing a disulfide pentagonal functional group in its side chain and its application. The polymer is based on obtaining a cyclocarbonate monomer containing a bifunctional bifunctional ring functional group through active controlled ring-opening polymerization, the molecular weight of which is controllable, the molecular weight distribution is relatively narrow and no protection and deprotection steps are required; The polymer obtained by the ring-opening polymerization of the cyclocarbonate monomer of the present invention has biodegradability and can be applied to the control of the drug release system, and the target tumor-targeted reductive-sensitive reversible crosslinked nanomaterial transporter to be produced is It supports the long-term circulation, but it accumulates highly in cancer cells and can rapidly release the drug in the cell, thereby killing cancer cells with high efficiency and specificity. At the same time, the biodegradable polymer has application prospects for tissue engineering supports and biochip coatings.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a carbonate polymer containing a disulfide pentagonal ring functional group in a side chain,

TECHNICAL FIELD The present invention relates to a biodegradable polymer material and its application, specifically to a carbonate polymer containing a disulfide pentagonal ring functional group in its side chain and its application, and belongs to the field of pharmaceutical materials.

The biodegradable polymer has a very unique performance. For example, it is usually biodegradable in the body with good biocompatibility, and the degradation products can be absorbed by the human body or discharged to the outside of the body through the normal physiological pathway of the human body, and thus can be used as a surgical suture, Materials, and drug controlled release transporters, and the like. Among them, synthetic biodegradable polymers are particularly attracted because their immunogenicity is comparatively low, and both performance including degradation performance and mechanical performance can be easily controlled. The synthetic biodegradable polymers mainly include aliphatic polyesters, polycarbonates, polyamino acids, polyphosphates, polyanhydrides, polyorthoesters, and the like. Of these, polycarbonates such as polytrimethylene carbonate (PTMC) and aliphatic polyesters such as polyglycolide (PGA), polylactide (PLA), lactide-glycolide copolymer (PLGA), polycaprolactone Polyester is the most commonly used biodegradable polymer and has already been approved by the US Food and Drug Administration (FDA).

However, conventional biodegradable polymers such as PTMC, PCL, PLA and PLGA have a relatively simple structure and lack functional groups that can be used for modification, so that a stable drug nanotransporter or stable surface modification coating layer It is difficult to provide.

Decomposition products of polycarbonate are mainly carbon dioxide and neutral diols, which do not produce acid decomposition products. Among them, the functional cyclic carbonate monomer can be copolymerized with a large number of cyclic ester monomers such as GA, LA and ε-CL, and other cyclic carbonate monomers to obtain biodegradable polymers with different performance.

In addition, in the prior art, in the ring-opening polymerization process, active groups in the cyclic carbonate monomer structure easily react, and therefore all the protection and deprotection steps are required when the functional polymer is prepared from the cyclic carbonate monomer, It makes it complicated.

It is an object of the present invention to provide a biodegradable polymer containing a disulfide pentagonal ring functional group in its side chain.

In order to accomplish the above object, a specific technical solution of the present invention is as follows.

A biodegradable polymer containing a disulfide pentagonal ring functional group in its side chain has one of the following chemical structural formulas;

Wherein R1 is selected from one of the following groups:

Wherein k = 20-250 and R4 is selected from one of the following groups;

R2 is selected from one of the following groups:

R3 is selected from one of the following groups:

또는

, 식 중 a=2, 3 또는 4이고; b=20-250이며;

or

, Wherein a = 2, 3 or 4; b = 20-250;

The molecular weight of the biodegradable polymer containing the disulfide pentagonal functional group in the side chain is 800 to 100000 Da.

In the above technical solution, the number of repeating units containing a disulfide pentagonal functional group in the molecular chain of the biodegradable polymer containing a disulfide pentagonal functional group in the side chain is 4 to 50.

The biodegradable polymer containing the disulfide pentagonal ring functional group in the side chain is obtained by ring-opening polymerization of a cyclocarbonate monomer containing a disulfide pentagonal functional group in a solvent in the presence of an initiator, or a cyclocarbonate containing a disulfide pentagonal functional group Obtained through ring-opening polymerization of monomers and other cycloester monomers, cyclocarbonate monomers; The other cyclocarbonate monomer includes trimethylene cyclocarbonate (TMC), and the other cyclic ester monomer includes caprolactone (? -CL), lactide (LA), or glycolide (GA).

The chemical structure of the cyclocarbonate monomer containing a disulfide pentagonal ring functional group in the side chain is as follows:

, 이는 이하 단계를 통해 제조될 수 있다.

, Which can be prepared via the following steps.

(1) Sodium dihydrogen sulphide (NaSH.H ₂ O) was dissolved in N, N-dimethylformamide (DMF), and dibromoneopentyl glycol was slowly added dropwise to a pressure-reducing dropping funnel. After completion of the reaction, the reaction product was distilled under reduced pressure to remove the solvent DMF, diluted with distilled water, extracted four times with ethyl acetate, and finally, the organic phase was concentrated by rotary evaporation to obtain yellow viscous compound A ;

The chemical structure of the compound A is as follows:

(2) preserving compound A in tetrahydrofuran solution and oxidizing in air for 24 hours to obtain compound B;

The chemical structure of the compound B is as follows:

(3) In a nitrogen gas atmosphere, compound B and ethyl chloroformate were dissolved in dried tetrahydrofuran. Then, triethylamine was slowly added dropwise through a pressure-reducing funnel, and the mixture was reacted in an ice water bath for 4 hours. , Filtration, rotary condensation of the filtrate, and recrystallization with ether three to five times to obtain a yellow crystal, i.e., a cyclocarbonate monomer containing a bifluoride pentane ring functional group.

The above-mentioned cyclocarbonate monomer can be subjected to ring-opening polymerization using bis (trimethylsilyl) amine zinc as a catalyst in polyethylene glycol as an initiator in dichloromethane to form a block polymer, and the reaction formula thereof is as follows:

The carbonate polymer containing the disulfide pentagonal ring functional group in the side chain has biodegradability and can produce nanoparticles (particle size 20-250 nm) and can further load anti-cancer drugs. Polymeric nanoparticles can catalyze stable chemical crosslinking or long-circulating by catalyzing a catalytic amount of a reducing agent such as dithiothreitol or glutathione, but after entering the cell, a large amount of reductant in the cell In the present environment, rapid decrosslinking occurs, releasing the drug and killing cancer cells with high efficiency. The first polymer produced by the present invention has good biocompatibility and, when applied as a drug transporter, increases the circulation time of the antitumor drug, increases the drug accumulation rate at the tumor site of the drug, It is possible to prevent damage, effectively kill tumor cells, and have a small effect on normal cells.

Accordingly, the present invention claims the protection of biodegradable polymers containing disulfide pentadecane functional groups in the side chain for application in the manufacture of drug release controlled vehicles; The molecular weight of the biodegradable polymer containing the disulfide pentagonal functional group in the side chain is 3,000 to 70,000 Da.

At the same time, after the biodegradable polymer containing the disulfide pentagonal ring functional group in the side chain forms a chemical crosslink to obtain a crosslinked nanoscale transporter, the surface of the crosslinked nanoscale transporter is coated with an RGD polypeptide, a nucleic acid plastomer, Lactose, and the like, so that the intake of the nanoparticles in cancer cells can be greatly increased.

The biodegradable polymer containing a disulfide pentagonal ring functional group in the side chain has biodegradability and can produce a biotissue support. The polymer can be prepared by polymerizing a polymer in the presence of a catalytic amount of a reducing substance such as dithiothreitol or glutathione Can be made into fibers through electrostatic spinning after reversible crosslinking. Such fibers can be adhered to the cells very well after modification, and the stability of the fibers is greatly enhanced through crosslinking, And it is possible to prevent clogging which is unstable in the support and is liable to dissociate. Accordingly, the present invention claims the protection of biodegradable polymers containing disulfide pentadecane functional groups in their side chains for their application in the manufacture of biostructured support materials. The molecular weight of the biodegradable polymer containing the disulfide pentagonal ring functional group in the side chain is 5,000 to 100,000 Da.

The present invention further claims the protection of the application of biodegradable polymers containing disulfide pentadecane functional groups to the side chains in the manufacture of biochip coating layers. The molecular weight of the biodegradable polymer containing the disulfide pentagonal ring functional group in the side chain is 800 to 10,000 Da. When the biodegradable polymer containing the disulfide pentagonal functional group in the side chain serves as a biochip coating layer, it is similar to a biotissue support and forms a stable chemical crosslinking by catalytic reduction of a catalytic amount of a reducing agent such as dithiothreitol or glutathione The biochip coating layer can be further stabilized in the body, thereby reducing non-specific adsorption and reducing the measurement noise of the biopsy content.

By the implementation of the above measures, the present invention has the following advantages in comparison with the prior art.

1. The present invention relates to a process for the preparation of a polymer having a molecular weight distribution which is capable of controlling molecular weight through the use of a cyclocarbonate monomer containing a disulfide pentagonal functional group in its side chain, A biodegradable polymer was obtained and the protection and deprotection steps in the prior art were not required in the polymerization process since the sulfur-sulfur halide group did not affect the ring opening polymerization of the cyclocarbonate monomer, simplifying the operation steps.

2. The biodegradable polymer containing the disulfide pentagonal ring functional group in the side chain disclosed by the present invention has an excellent biodegradability and can be applied to the control of the drug release system and can be used as a tumor targeting oriented reduction sensitive reversible crosslinked nanomaterial transporter Can support the long-term circulation in the body, and the cancer cells can be rapidly crosslinked in highly concentrated cells to release the drug, thereby killing cancer cells specifically and efficiently.

3. The cyclocarbonate monomer disclosed by the present invention can be easily prepared and can obtain a biodegradable polymer containing a disulfide pentagonal functional group in its side chain through easy ring-opening polymerization; The polymer can also be self-assembled and can be applied to drug release system control, tissue engineering and biochip coating layers, and has good application value in terms of biomaterials.

1 is a hydrogen nuclear magnetic resonance spectrum diagram of a polymer PEG5k-P (CDC2.5k- co- C3.9k) in Example 2. Fig.
2 is a nuclear magnetic resonance spectrum diagram of Polymer P (CDC- co- CL) (6.21k) -PEG (0.5k) -P (CDC- co- Cl) (6.21k) in Example 15.
3 is a particle size distribution diagram of the polymer micelle nanoparticles PEG5k- b- PCDC2.8k in Example 16. Fig.
Fig. 4 is a graph showing the particle diameter of the crosslinked micelle nanoparticles PEG5k- b- PCDC2.8k in a highly diluted state in Example 17. Fig.
Fig. 5 is a graph showing the particle diameter of crosslinked micellar nanoparticles PEG5k- b- PCDC2.8k in the presence of glutathione, which is a reducing material in Example 17. Fig.
FIG. 6 is a toxicity profile of Raw264.7 and MCF-7 cells of crosslinked micellar nanoparticles PEG5k- b- PCDC2.8k in Example 17. FIG.
Fig. 7 is a result of in vitro release of DOX transport crosslinked micelle nanoparticles PEG5k- b- PCDC2.8k in Example 18. Fig.
8 is the toxicity of Raw264.7 and MCF-7 cells of DOX transport crosslinked micellar nanoparticles PEG5k- b- PCDC2.8k in Example 18. FIG.
9 is a particle size distribution and a projection electron micrograph of cross-linked polymeric vesicles nanoparticles PEG5k-P (CDC4.9k- co- TMC19k) in Example 19. FIG.
10 is the toxicity profile of the target cross-linked vesicle nanoparticles cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k) / PEG5k-P (CDC4.9k- co- TMC19k) in Example 19 to U87MG cells.
11 is the toxicity of the target cross-linked vesicle nanoparticles loaded with DOX in Example 19 to U87MG cells.
12 is a blood circulation diagram in a mouse body of DOX-loaded crosslinked nanoparticles PEG5k- b- PCDC 2.8k of Example 20. Fig.
13 is a biodistribution result of melanoma mouse of DOX-loaded crosslinked nanoparticle PEG5k- b- PDC2.8k of Example 21.
14 is a diagram of a result of the treatment of Example 22, DOX loading PEG5k- b -PCDC2.8k cross-melanoma mouse of nanoparticles.
15 is a blood circulation diagram in a mouse body of DOX-loaded target cross-linked vesicles of Example 23. Fig.
16 is a biodistribution diagram of the human brain malignant glioma mouse of DOX-loaded target cross-linked vesicles in Example 24.
FIG. 17 is a therapeutic effect diagram of DOX-loaded target cross-linked vesicles of Example 25 for human brain malignant glioma mice.
Figure 18 is a therapeutic effect of the DOX loaded target cross-linked vesicles of Example 26 on melanoma mice.
Fig. 19 is a biodistribution diagram of DOX-loaded target cross-linked vesicles of Example 27 in a lung cancer mouse body. Fig.
20 is a TEM diagram of the surface-modified gold nanorods of PEG5k-PLGA 7.8k-PCDC 1.7k in Example 28. FIG.
FIG. 21 is a photograph of the polymer PCL and P (CDC0.8k- co- CL92k) in Example 30 after film formation and immersion in physiological saline for 2 weeks.

BRIEF DESCRIPTION OF THE DRAWINGS Fig.

Example 1 : Synthesis of cyclocarbonate monomer (CDC) containing a bifunctional bifunctional ring functional group

1. Monohydrate sodium hydrosulfide (28.25 g, 381.7 mmol) was dissolved in 400 mL of N, N-dimethylformamide (DMF) and heated to completely dissolve at 50 째 C followed by dibromoneopentyl glycol , 76.4 mmol) was gradually added dropwise and reacted for 48 hours. The reaction product was distilled under reduced pressure to remove the solvent DMF, diluted with 200 mL of distilled water, extracted four times with 250 mL of ethyl acetate, and finally, the organic phase was rotary evaporated to obtain yellow viscous compound A. The yield was 70%.

2. Compound A dissolved in 400 mL of tetrahydrofuran (THF) was left in the air for 24 hours to oxidize the intermolecular sulfhydryl to a sulfur-sulfur bond to obtain compound B, yield> 98 %to be.

3. Under nitrogen gas protection, compound B (11.7 g, 70.5 mmol) is dissolved in dry THF (150 mL) and stirred until completely dissolved. Was then added to, ethyl chloroformate (15.65mL, 119.8mmol) was cooled to 0 ℃ dropwise _{Next, Et 3 N (22.83mL, 120.0mmol} ). After the addition is complete, the system is allowed to react for 4 h continuously under ice-bath bath conditions. After completion of the reaction, the resulting Et ₃ N HCl was filtered, the filtrate was concentrated by rotary evaporation, and finally recrystallized with ether several times to obtain a yellow crystal, namely, a cyclocarbonate monomer (CDC) containing a disulfide pentagonal ring- , And the yield is 64%.

Example 2 : Synthesis of polymer PEG5k- b- PCDC2.8k containing a disulfide pentagonal ring functional group in the double-block side chain

In a nitrogen gas environment, 0.3 g (1.56 mmol) of CDC monomer and 2 mL of dichloromethane were charged into a sealed reactor, and then 0.5 g (0.1 mmol) of a polyethylene glycol having a molecular weight of 5000 and 1 mL of a catalyst bis (bistrimethylsilyl) A zinc dichloride solution (0.1 mol / L) of zinc amine was added, the reactor was sealed, transferred to a glove box, and reacted in an oil bath at 40 ° C for one day. The reaction was then terminated with glacial acetic acid and precipitated in cold ether After final filtration and vacuum drying, the product PEG5k- b- PCDC2.8k is obtained.

^{1 H NMR (400 MHz, CDCl} 3): 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 4.05 (s, -CH 2 OCOCHCH 2 -), 4.07 (s, -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2).

In the formula, m = 113.6 and n = 14.6.

Example 3 : Synthesis of Polymer PEG5k-P (CDC2.5k-co-CL3.9k) Containing a Disulfide Bivalent Ring on a Double Block Side Chain

In a nitrogen gas environment, 0.28 g (1.46 mmol) of CDC monomer and 0.4 g (3.51 mmol) of caprolactone (ε-CL) were dissolved in 3 mL of dichloromethane and placed in a sealed reactor, 0.5 g (0.1 mmol) of polyethylene glycol and a dichloromethane solution (0.1 mol / L) of 0.1 mol / L of catalytic bis (bistrimethylsilyl) amine zinc were charged. Then, the reactor was sealed and transferred to a glove box, The reaction was terminated with glacial acetic acid, precipitated in cold ether, and finally filtered and vacuum dried to obtain the product PEG5k-P (CDC2.5k-co-CL3.9k). GPC measurement Molecular weight: 14.0 kDa, molecular weight distribution: 1.56.

In the formula, m = 113.6, x = 34.2, y = 13.0, n = 47.2.

1 is a nuclear magnetic resonance spectrum diagram of the polymer. ^{1 H NMR (400 MHz, CDCl} 3): 1.40 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 1.65 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 2.30 (t _{, -COCH 2 CH 2 CH 2 CH} 2 CH 2 -), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 4.03 (t, -COCH 2 CH 2 CH 2 CH 2 CH 2 O- _{), 4.05 (s, -CH 2} OCOCHCH 2 -), 4.07 (s, -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2).

Example 4 : Synthesis of Polymer PEG5k-P (CDC3.8k- co- C14k) Containing Disulfide Bifunctional Ring in the Double Block Side Chain

In a nitrogen gas environment, 0.5 g (2.6 mmol) of CDC monomer and 1.5 g (13.2 mmol) of caprolactone (ε-CL) were dissolved in 10 mL of dichloromethane and placed in a sealed reactor, A dichloromethane solution (0.1 mol / L) of 0.5 g (0.1 mmol) of polyethylene glycol and 1 mL of catalytic bis (bistrimethylsilyl) amine zinc was charged, and then the reactor was sealed and transferred to a glove box. After reacting for one day, the reaction is terminated with glacial acetic acid, precipitated in cold ether, and finally filtered and vacuum dried to obtain the product PEG5k-P (CDC3.8k- co- Cl14k). GPC measurement Molecular weight: 30.6 kDa, molecular weight distribution: 1.34

In the formula, m = 113.6, x = 122.8, y = 19.8, n = 142.

Example 5 : Synthesis of Polymer PEG 1.9k-P (CDC3.9k- co- C3.8k) Containing a Disulfide Bivalent Ring on a Double Block Side Chain

In a nitrogen gas environment, 0.4 g (2.1 mmol) of CDC monomer and 0.4 g (3.51 mmol) of caprolactone (ε-CL) were dissolved in 3 mL of dichloromethane and placed in a sealed reactor, A dichloromethane solution (0.1 mol / L) of 0.4 g (0.21 mmol) of polyethylene glycol and 1 mL of catalytic bis (bistrimethylsilyl) amine zinc was introduced. Then, the reactor was sealed and transferred to a glove box. After the reaction for one day, the reaction is terminated with glacial acetic acid, precipitated in cold ether, and finally filtered and vacuum dried to obtain the product PEG1.9k-P (CDC3.9k- co- C3.8k). GPC measured molecular weight: 0.96 kDa, molecular weight distribution: 1.35.

In the formula, m = 43.2, x = 33.3, y = 20.3, n = 53.6.

Example 6 Synthesis of Homopolymer Alk-PCDC 2.8k Containing Disulfide Bifunctional Functional Group on the Side Chain

In a nitrogen gas environment, 0.3 g (1.6 mmol) of CDC monomer was dissolved in 1 mL of dichloromethane, placed in a sealed reactor and then treated with 1 mmol / L of purified propargyl alcohol and 1 mL of catalytic bis (bistrimethylsilyl) amine A dichloromethane solution of zinc (0.1 mol / L) was added, the reactor was sealed, transferred to a glove box, and reacted in an oil bath at 40 ° C for one day. After completion of the reaction with glacial acetic acid, , Finally filtered and vacuum dried to obtain the product Alk-PCDC 2.8k.

Example 7 Synthesis of Carbonate Polymer iPr-P (CDC0.8k- co- CL92k) Containing Disulfide Bifunctional Functional Group on the Side Chain

In a nitrogen gas environment, 0.1 g (0.52 mmol) of CDC monomer and 10 g (87.7 mmol) of caprolactone monomer (CL) were dissolved in 10 mL of ε-CL in dichloromethane and placed in a sealed reactor, followed by the addition of 6 mg of isopropanol (0.1 mol / L) of zinc bis (trimethylsilyl) amine catalyst and 1 mL of a catalytic amount of zinc bis (trimethylsilyl) amine were introduced into a glove box. Then, the reactor was sealed and transferred to a glove box, The product iPr-P (CDC-co-CL) (0.8k-92k) was obtained by final filtration and vacuum drying after completion of the reaction with glacial acetic acid and precipitation in cold ether. 102.3 kDa, molecular weight distribution: 1.36

In the formula, x = 4.2, y = 80.7, n = 84.9.

Example 8 : Synthesis of polymer PEG5k-PCDC1.0k-PCL3.2k containing a disulfide bifunctional ring in the triple block side chain

In a nitrogen gas environment, 0.12 g (1.5 mmol) of CDC monomer was dissolved in 2 mL of dichloromethane and charged to the sealed reactor. Then 0.5 g (0.31 mmol) of polyethylene glycol having a molecular weight of 5000 and 1 mL of catalyst bis (0.1 mol / L) of zinc chloride was added to the reactor, and the reactor was sealed and transferred to a glove box. The reactor was reacted for one day in an oil bath at 40 DEG C, and then, in a glove box, The reaction was terminated with glacial acetic acid and precipitated in cold ether. Finally, filtration and vacuum drying were performed to obtain the product, tris (3-hydroxyphenyl) Obtain polymer PEG5k-PCDC 1.0k-PCL 3.2k. GPC measurement Molecular weight: 10.4 kDa, molecular weight distribution: 1.45.

^{1 H NMR (400 MHz, CDCl} 3): 1.40 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 1.65 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 2.30 (t _{, -COCH 2 CH 2 CH 2 CH} 2 CH 2 -), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 4.03 (t, -COCH 2 CH 2 CH 2 CH 2 CH 2 O- _{), 4.05 (s, -CH 2} OCOCHCH 2 -), 4.07 (s, -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2).

Example 9 Synthesis of PEG5k-P (CDC3.2k- co- TMBPEC3.5k) Containing a Disulfide Bivalent Ring on a Double Block Side Chain

In a nitrogen gas environment, 0.4 g (2.1 mmol) of CDC monomer and 0.4 g (1.2 mmol) of 2,4,6-trimethoxybenzylidene pentaerythritolcarbonate monomer (TMBPEC) were dissolved in 5 mL of dichloromethane, (0.1 mmol) of a polyethylene glycol having a molecular weight of 5000 and a dichloromethane solution (0.1 mol / L) of zinc catalytic bis (bistrimethylsilyl) amine were introduced into the reactor, and then the reactor was sealed After the reaction was completed with glacial acetic acid, the reaction was terminated, precipitated in cold ether, and finally filtered and vacuum-dried to obtain the product PEG5k-P (CDC3.2k- co- TMBPEC3.5k). GPC measurement Molecular weight: 12.4 kDa, molecular weight distribution: 1.47.

In the formula, m = 113.6, x-16.7, y = 10.2, n = 26.9.

Example 10 : Synthesis of PEG 1.9k-PCL 1.8k-PCDC 0.7k containing a disulfide pentadecane ring functional group in a triblock side chain

In a nitrogen gas environment, 0.2 g (1.76 mmol) of caprolactone (? -CL) was dissolved in 2 mL of dichloromethane and charged into a sealed reactor. Then 0.19 g (0.1 mmol) of polyethylene glycol with a molecular weight of 1900 and 0.1 a dichloromethane solution (0.1 mol / L) of zinc bis (trimethylsilyl) amine catalyst / bis (trimethylsilyl) amine / mol was added to the reactor, and then the reactor was sealed and transferred to a glove box. 80 mg (0.42 mmol) of CDC monomer was added to the glove box under nitrogen gas protection, and the reaction was continued for one day. The reaction was terminated with glacial acetic acid and precipitated in cold ether. Finally, filtration and vacuum drying were conducted to obtain a triblock polymer PEG1.9k-PCL1.8k-PCDC0.7k. GPC measured molecular weight: 0.64 kDa, molecular weight distribution: 1.32.

^{1 H NMR (400 MHz, CDCl} 3): 1.40 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 1.65 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 2.30 (t _{, -COCH 2 CH 2 CH 2 CH} 2 CH 2 -), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 4.03 (t, -COCH 2 CH 2 CH 2 CH 2 CH 2 O- _{), 4.05 (s, -CH 2} OCOCHCH 2 -), 4.07 (s, -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2).

Example 11 Synthesis of Polymer PEG5k-P (CDC4.9k- co- TMC19k) Containing a Disulfide Bivalent Ring on a Double Block Side Chain

In a nitrogen gas environment, 0.1 g (0.52 mmol) of CDC monomer and 0.4 g (3.85 mmol) of trimethylene carbonate (TMC) were dissolved in 3 mL of dichloromethane, charged into a sealed reactor, 0.1 mol (0.02 mmol) of a glycol and 0.1 mol / L of a dichloromethane solution (0.1 mol / L) of a catalytic bis (bistrimethylsilyl) amine zinc were introduced into the reactor. Then, the reactor was sealed and transferred to a glove box, After reacting in an oil bath for one day, the reaction was terminated with glacial acetic acid, precipitated in cold ether, and finally filtered and vacuum dried to obtain a double-block polymer PEG5k-P (CDC4.9k-co-TMC19.0k) . GPC measurement Molecular weight: 34.5 kDa, molecular weight distribution: 1.48.

^{1 H NMR (400 MHz, CDCl} 3): 2.08 (t, -COCH 2 CH 2 CH 2 O-), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 3.65 (t, -O CH ₂ CH ₂ O-), 4.28 (t, -COCH ₂ CH ₂ CH ₂ O-), 4.31 (m, -CCH ₂ ).

In the formula, m = 113.6, x = 25.5, y = 186.3, n = 211.8.

Example 12 : Synthesis of a target polymer iRGD-PEG6k-P (CDC4.8k- co- TMC19.2k) containing a bifunctional bifunctional ring on the double-block side chain modified with iRGD polypeptide

The synthesis of the polymer iRGD-PEG6k-P (CDC4.8k- co- TMC19.2k) is divided into two stages. The synthesis of the first maleimide functionalizing polymer Mal-PEG6k-P (CDC4.8k- co- TMC19.2k) was the same as that of Example 11, except that mPEG having a molecular weight of 5000 was replaced with Mal-PEG having a molecular weight of 6000 Da Except that it initiates polymerization. ^{1 H NMR (400 MHz, CDCl} 3): 2.08 (t, -COCH 2 CH 2 CH 2 O-), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 3.65 (t, -O CH ₂ CH ₂ O-), 4.28 (t, -COCH ₂ CH ₂ CH ₂ O-), 4.31 (m, -CCH ₂ ), 6.70 (s, Mal). GPC measured molecular weight: 38.6 kDa, molecular weight distribution: 1.42.

In the formula, m = 136.4, x = 24.8, y = 188.4, and m = 213.2.

The second step is the Michael addition of the polypeptide iRGD and the obtained polymer. The polymer Mal-PEG6k-P (CDC4.8k- co- TMC19.2k) was first dissolved in DMF, the PB buffer solution was added dropwise to the nanoparticles, the organic solvent was removed by dialysis, and then a 2-fold molar amount of iRGD After 2 days of reaction at 30 ° C, unbound iRGD is removed by dialysis and lyophilized to obtain the final product iRGD-PEG6k-P (CDC 4.8k- co- TMC19.2k). The NMR and BCA protein kidd calculations show that the iRGD graft rate is 92%.

Example 13 : Synthesis of target polymer cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k) containing a bifunctional bifunctional ring on the double-block side chain modified with cRGD polypeptide

The synthesis of polymer cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k) is similar to Example 12 and is divided into two stages. The synthesis of the first N-hydroxysuccinimide modified polymer NHS-PEG6k-P (CDC4.6k- co- TMC18.6k) is similar to that of Example 11, except that mPEG having a molecular weight of 5000 Da is replaced with 6000 Da NHS-PEG to initiate polymerization. ^{1 H NMR (400 MHz, CDCl} 3): 2.08 (t, -COCH 2 CH 2 CH 2 O-), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 3.65 (t, -O CH ₂ CH ₂ O-), 4.28 (t, -COCH ₂ CH ₂ CH ₂ O-), 4.31 (m, -CCH ₂ ), 2.3 (s, NHS). GPC measurement Molecular weight: 37.6 kDa, molecular weight distribution: 1.38.

In the formula, m = 136.4, x = 24.0, y = 178.8, and n = 202.8.

The second step is to bind the polypeptide cRGD and the obtained polymer through an amidation reaction. First, the polymer was dissolved in DMF, and 2-fold molar amount of cRGD was added. After reacting at 30 캜 for 2 days, unbound glass cRGD was removed by dialysis and freeze-dried to obtain the final product cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k). The calculation of NMR and BCA protein kid shows that the cRGD graft rate is 88%.

Example 14 : Synthesis of Polymer PEG5k-PLA7.8k-PCDC 1.7k Containing a Disulfide Bifunctional Ring in the Trimeric Block Side Chain

In a nitrogen gas environment, 0.45 g (3.13 mmol) of lactide (LA) was dissolved in 3 mL of dichloromethane and charged into a sealed reactor. Then 0.25 g (0.05 mmol) of polyethylene glycol with a molecular weight of 5000 and 1 mL of catalyst A dichloromethane solution of bis (trimethylsilyl) amine zinc (0.1 mol / L) was added, and then the reactor was sealed and transferred to a glove box. The reactor was reacted for one day in an oil bath at 40 ° C., The reaction was terminated with glacial acetic acid, precipitated in cold ether, and finally filtered and vacuum-dried to obtain a triblock polymer PEG5k-PLA7.8k (0.52mmol) -PCDC1.7k. GPC measurement Molecular weight: 16.8 kDa, molecular weight distribution: 1.47.

^{1 H NMR (400 MHz, CDCl} 3): 1.59 (m, -COCH (CH 3) O-), 3.08 (s, -CCH 2), 3.30 (m, -OCH 3), 3.65 (m, -O CH _{2 CH 2 O-), 4.07 (} s, -O CH 2 CCH 2 O-), 5.07 (m, -CO CH (CH 3).

In the formula, m = 113.6, x = 122.2, y = 8.9, n = 131.1.

Example 15 : Synthesis of Polymer P (CDC- co- CL) (6.21k) -PEG (0.5k) -P (CDC- co- L) (6.21k) containing a disulfide pentacyano ring in the triblock side chain

In a nitrogen gas environment, 1.5 g (13.2 mmol) of epsilon-CL and 0.0625 g (0.325 mmol) of CDC monomer were dissolved in 8 mL of dichloromethane and placed in a sealed reactor, then 0.05 g of PEG500 (0.01 mmol) (0.1 mol / L) of zinc bis (trimethylsilyl) amine catalyst and 1 mL of catalytic bis (trimethylsilyl) amine zinc were added and reacted for one day. The reaction was terminated with glacial acetic acid, precipitated in cold ether, via the vacuum-drying to obtain the triblock polymer P (CDC-co-CL) (6.21k) -PEG (0.5k) -P (CDC- co -CL) (6.21k). GPC measurement Molecular weight: 14.6 kDa, molecular weight distribution: 1.38.

2 is a nuclear magnetic resonance spectrum diagram of the polymer. ^{1 H NMR (400 MHz, CDCl} 3): 1.40 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 1.65 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 2.30 (t _{, -COCH 2 CH 2 CH 2 CH} 2 CH 2 -), 3.08 (s, -CCH 2), 4.03 (t, -COCH 2 CH 2 CH 2 CH 2 CH 2 O-), 4.05 (s, -CH 2 _{OCOCHCH 2 -), 4.07 (s} , -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2).

In the formula, m = 11.4, x = 6.3, y = 43.9, n = 51.2.

From the above results, it can be seen that, through characterization of a series of polymers, the ring-opening polymerization and copolymerization of CDC can be controlled, the molecular weight thereof is compatible with the design, and the molecular weight distribution of the polymer is relatively narrow.

Example 16 : Preparation of polymer micelle nanoparticles PEG5k- b- PCDC 2.8k

Polymer nanoparticles are prepared by dialysis. DMF was added dropwise a solution of PEG5k- b -PCDC2.8k of 200μL (2mg / mL) in phosphate buffer solution of 800μL (10mM, pH 7.4, PB ) , and put in the dialysis bag (MWCO 3500) and dialyzed for one night, the water And the dialysis medium is PB (10 mM, pH 7.4). The size of the obtained micellar nanoparticles is 173 nm as measured by a dynamic light scattering particle size analyzer (DLS), and the particle size distribution is very narrow. See FIG.

Example 17 : Crosslinking, De-crosslinking, and Cytotoxicity of Polymer Micelle Nanoparticles PEG5k- b- PDC2.8k

Nitrogen gas was injected into the aqueous solution of micellar nanoparticles for 20 minutes to rapidly purify the air. Then, 10 μL of the nanoparticle solution (1 mL, 0.25 mg / mL, 3.21 × 10 ^-5 mmol) Dithiothreitol (DTT) (0.007 mg, 4.67 × 10 ^-5 mmol, 10% of the number of moles of the functional group of the lipoic acid functional group) was added thereto, and the mixture was reacted for one day with stirring at room temperature. One day after dialysis, the particle size was measured to be 150 nm, which was about 15% smaller than the non-crosslinked particle size. After the crosslinking, the nanoparticle concentration was diluted below the CMC, and the particle size and particle size distribution showed almost no change. It is stable under physiological conditions, indicating that the disulfide bridging can improve the stability of the nanoparticles to a considerable extent. Please refer to Fig.

Disulfide bonds are very easy to insulate under the action of reducing agents such as glutathione (GSH) or DTT. Nitrogen gas was introduced into the crosslinked nanoparticle solution for 10 minutes under nitrogen gas protection and 37 ° C, GSH was added thereto so that the final concentration in the solution of the micellar nanoparticles was 10 mM, and the number of crosslinked nanoparticles Track changes. As shown in FIG. 5, after the addition of 10 mM reduced GSH, the particle diameter of the crosslinked nanoparticles was gradually destroyed with the passage of time, and the disulfide ring of the polymer was insulated in a state where a large amount of reducing substance was present . Since there is a high concentration of GSH in the cytoplasm, the produced nanoparticles are stable in circulation but release quickly after being endocytosed by monocytes.

The cytotoxicity of the crosslinked nanoparticles was tested using the MTT method. The cells used were MCF-7 (human mammary carcinoma cells) and Raw 264.7 (mouse macrophage) cells. HeLa or Raw 264.7 cells were inoculated in a 96-well plate at a concentration of 1 × 10 ⁴ cells / mL in a volume of 100 μL per well. After culturing the cells until they adhered to the wall, a culture solution containing micellar nano- And the cell-free control wells and the culture medium-treated wells (four parallel wells) were provided separately. After culturing for 24 hours, a 96-well plate was taken out, and 10 μL of MTT (5.0 mg / mL) was added. After the incubation was continued for 4 hours, 150 μL of DMSO was added to each well to dissolve the generated crystals. , The absorbance value (A) was measured at the 492 nm region, and the cell viability was calculated by adjusting the no-treatment well to 0.

In the equation, A _T is the absorbance at the 492 nm region of the experimental group and Ac is the absorbance at the 492 nm region of the untreated control group. The polymer concentrations are 0.1, 0.2, 0.3, 0.4 and 0.5 mg / mL, respectively. Figure 6 shows the cytotoxicity results of the nanoparticles. As can be seen from the figure, when the concentration of the micelle nanoparticles is increased from 0.1 to 0.5 mg / mL, the survival rate of Raw264.7 and MCF-7 cells is still 85% It was higher, which demonstrated that the PEG5k- b -PCDC2.8k micellar nanoparticle has a good biocompatibility.

Example 18 : Drug loading of crosslinked nanoparticles PEG5k- b- PCDC2.8k, in vitro release and cytotoxicity

With doxorubicin (DOX) as a drug, the overall operation was carried out under light-shielding conditions. First, the hydrochloride of doxorubicin is removed, and the operation is as follows. 1.2 mg (0.002 mmol) of DOX are dissolved in 225 μL of DMSO, 0.58 mL (m = 0.419 mg, 0.004 mmol) of triethylamine is added and stirred for 12 hours, and the supernatant is decanted off. The DMSO solution concentration of DOX is 5.0 mg / mL. PEG5k- b- PCDC2.8k was dissolved in DMF and mixed well with the DMSO solution of DOX according to the mass ratio of the drug and the polymer. Then, the second order water, which was four times its volume, was added in the stirred state, Dialyzed in water.

Crosslinking of the drug loading nanoparticles was also carried out according to the crosslinking method of Example 17. 100 μL of the cross-linked DOX-loaded micellar nanoparticle solution was freeze-dried and then dissolved in 3.0 mL of DMSO, tested using a fluorescence spectrophotometer, and the incorporation rate was calculated by combining with the standard curve of DOX.

Drug loading (DLC) and filling rate (DLE) are calculated according to the following formula.

Drug loading (wt.%) = (Drug weight / polymer weight) x 100

Encapsulation rate (%) = (drug loaded weight / total drug dose) × 100

Table 1 shows a mopping that the loading operation of high efficiency for the loading of the PEG5k- b -PCDC2.8k micellar nanoparticle DOX.

Results of drug loading and inclusion rate in crosslinked polymer nanoparticles loaded with doxorubicin polymer Feed ratio (wt.%) Drug loading (wt.%) Filling rate (%) Size (nm) Particle size distribution
PEG5k-b-PCDC 2.8k
5 4.0 83.3 150.3 0.17 10 7.4 80.0 162.1 0.22 15 9.1 68.2 173.2 0.19

The in vitro release experiments of DOX were shaken (200 rpm) at 37 ° C on a constant temperature shaker and there were two parallel samples for each group. The first group is the release group in PB (10 mM, pH 7.4) mimicking the intracellular reducing environment of 10 mM GSH of DOX loaded crosslinked micellar nanoparticles; The second group is the release group of PBX (10 mM, pH 7.4) of DOX loaded crosslinked micellar nanoparticles. The concentration of the drug-loaded micelle nanoparticles is 25 mg / L. Take 0.5 mL of the drug-loaded micellar nanoparticles into a dialysis bag (MWCO: 12,000-14,000), add 25 mL of the dialysis solvent corresponding to each test tube, And tested with a dialysis bag external medium, while 5.0 mL of the corresponding medium was replenished to the test tube. The drug concentration in the solution was measured using an EDINBURGH FLS920 fluorescence photometer. Figure 7 shows the relationship between cumulative doses of DOX and time. As shown in the figure, after the introduction of GSH into the tumor cells, the release was significantly faster than the sample without GSH, Demonstrate that nanoparticles can effectively release the drug in the presence of 10 mM GSH.

The toxicity of PEG5k- b -PCDC2.8k crosslinked nanoparticles loaded with DOX by the MTT method to mouse macrophages Raw264.7 and human mammary carcinoma cells MCF-7 was tested, and drug-loaded microsaccharide micellar nanoparticles and free drugs were added to the control . For Raw264.7 cells, Raw264.7 cells were inoculated in a 96-well plate at a density of 1 × 10 ⁴ cells / mL in a volume of 100 μL per well, cultured until the cells adhered to the wall, , 1, 5, 10, 50 and 100 μg / mL of DOX-loaded crosslinked nanoparticle solution and fresh DOX-containing culture medium were added and cultured for 48 hours in an incubator. mL) was added thereto, followed by incubation for 4 hours. Then, 150 μL of DMSO was added to each well to dissolve the resulting crystals. The absorbance value (A) was measured at 492 nm with a microplate reader. Was adjusted to 0 to calculate cell viability.

FIG. 8 shows the toxicity of the drug-loaded crosslinked micellar nanoparticles PEG5k- b- PCDC2.8k to Raw264.7 and MCF-7 cells, showing the half-life of Raw264.7 cells of DOX- The concentration is 4.89 μg / mL, and the half-life concentration for MCF-7 cells is 2.31 μg / mL. Therefore, the DOX-loaded crosslinked nanoparticles can effectively release drug in cells and kill cancer cells.

Example 19 : Formation of cross-linked polymeric vesicles nanoparticles PEG5k-P (CDC4.9k- co- TMC19k), biocompatibility, toxicity to MCF-7, U87-MG and A549 cells of drug loaded cross-

Similar to Example 16, the polymer PEG5k-P (CDC4.9k- co- TMC19k) can form nanoparticles and, as can be seen from tests such as TEM and CLSM, the structure is a polymer vesicle structure, 9. As can be seen from FIG. 9A, the nanobesecle formed by the DLS test is about 100 nm and the particle size distribution is very narrow. As shown in FIG. 9B, the vesicle measured by TEM is a hollow structure. Similar to Example 17, the obtained vesicles are capable of cross-linking and also have a reduced susceptibility to degradation, and toxicity tests on MCF-7 human mammary tumor cells, U87MG human brain glioma cells and A549 lung cancer cells are conducted Lt; / RTI > 10, when the vesicle concentration was increased from 0.3 mg / mL to 1.5 mg / mL, the survival rate of MCF-7, U87MG and A540 cells after incubation for 24 hours was still 85% -110% It is explained that the nanobesecycles have good biocompatibility. See Figure 11 for toxicity to U87MG human brain glioma of the cRGD target drug loading crosslinked vesicle. As can be seen in the figure, when the target cRGD polymer weight ratio was increased from 10% to 30%, the half-life rate was reduced from 3.57 μg / mL to 1.32 μg / mL, close to or lower than the free drug, Was 2.3 to 6.4 fold lower than the drug-loaded crosslinked vesicles.

The loading of DOX · HCl is carried out by the pH gradient method, and the hydrophilic DOX is encapsulated using the point where the pH inside and outside the vesicle is different. The drug loaded crosslinked vesicles were prepared according to different dose ratios of 10% -30%, and the diameters of the crosslinked vesicles measured via DLS after removal of unencapsulated free drug by dialysis were 105-124 nm, The particle size distribution was still very narrow, 0.10-0.15, and the sealing efficiency against hydrophilic DOX was very high (63% -77%).

Example 20 : Measurement of blood circulation in mouse body of drug-loaded PEG5k- b -PCDC2.8k crosslinked nanoparticles

C57BL / 6 black mice (laboratory animal center, Shanghai Institute of Bioscience, China Academy of Sciences) were selected for body weight of 18 ~ 20g and 4 ~ 6 weeks old. After body weight was measured, groups were evenly divided and drug loading nanoparticle drug And free drug were injected into the mouse body via the tail vein. The dose of DOX was 10mg / kg, and approximately 10 μL of blood was collected at fixed time points of 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours. After precise calculation, blood was extracted by adding Triton (1%) and 500 μL of DMF (containing 20 mM DTT and 1 M HCl) at a concentration of 100 μL, centrifuged (20000 rpm, 20 minutes) The supernatant was removed and the amount of DOX at each time point was measured with a fluorescence photometer.

12 is a circulation of the mouse body of DOX loaded cross-linked nanoparticles PEG5k- b -PCDC2.8k FIG. The abscissa is the time point, and the ordinate is the amount of DOX (ID% / g) in the blood per gram of total DOX injection. As can be seen from the figure, the circulation time of DOX is very short and it is very difficult to detect DOX already in 2 hours, while the crosslinked nanoparticles were still 4 ID% / g after 24 hours. The half-life of the crosslinked nanoparticles in the mouse can be calculated to be 4.67 hours, while the DOX is only 0.21 hours. Therefore, the drug-loaded crosslinked nanoparticles are stable in the mouse body and have a relatively long circulation time.

Example 21 : Biodistribution of drug-loaded PEG5k- b- PDC2.8k crosslinked nanoparticles to melanoma mice

C57BL / 6 black mice (experimental animal center, Shanghai Institute of Life Science, China Academy of Sciences) were selected for body weight of 18 ~ 20g and 4-6 weeks old. After body weight was measured, groups were divided into groups of 1 × 10 ⁶ B16 melanoma cells were subcutaneously injected and approximately two weeks later, when tumor size reached 100-200 mm ³ , drug-loaded nanoparticles and DOX were injected intravenously into the mouse via tail vein (dose of DOX 10 mg / kg), 6, 12 and 24 hours later, the mice were sacrificed and the tumor and heart, liver, spleen, lung and kidney tissues were harvested and washed and weighed, then 500 μL of 1% Triton was added and homogenized And then extracted with 900 mu L of DMF (containing 20 mM of DTT and 1 M of HCl). After centrifugation (20,000 rpm, 20 minutes), the supernatant was removed, and the amount of DOX at each time point was measured with a fluorescence photometer.

13 is a biodistribution result of melanoma mouse of DOX loading crosslinked nanoparticle PEG5k- b- PCDC 2.8k. The abscissa is the tissue organs, and the ordinate is the amount of DOX (ID% / g) in the tumor or tissue per gram of total DOX injection. The tumor accumulation amounts of drug-loaded nanoparticles during 3, 6, 12 and 24 hours were 3.12, 2.93 and 2.52 ID% / g, respectively, which were 3 to 12 times higher than those of DOX 1.05, 0.52 and 0.29 ID% Loading crosslinked nanoparticles accumulate relatively large amounts in the tumor area through the EPR effect and also have a relatively long sustainable time.

Example 22 : Therapeutic experiment on melanoma mouse of drug-loaded PEG5k- b- PDCD2.8k crosslinked nanoparticles

C57BL / 6 black mice (experimental animal center, Shanghai Institute of Life Science, China Academy of Sciences) were selected for body weight of 18 ~ 20g and 4-6 weeks old. After body weight was measured, groups were divided into groups of 1 × 10 ⁶ B16 black tumor cells were subcutaneously injected and approximately one week later, when tumor size reached 30-50 mm ³ , drug loaded nanoparticles and DOX were injected into the mouse body via the tail vein on days 0, 2, 4, 6 and 8 Lt; / RTI > The doses of DOX in the drug-loaded nanoparticles were 10, 20, and 30 mg / kg, and the dose of DOX was 10 mg / kg. The body weight of each group of mice was measured daily for 0 to 15 days. The volume was measured accurately. The tumor volume calculation method is V = (L × W × H) / 2, where L is the length of the tumor, W is the width of the tumor, and H is the thickness of the tumor. Survival of the mice was continuously monitored for up to 46 days.

Figure 14 is a diagram DOX treatment results for melanoma mouse of the loaded PEG5k- b -PCDC2.8k crosslinked nano-particles, of which Fig. A curve is suppressed tumor growth, Figure B is a weight variation curve of mice, also C Is the survival curve of the mouse. 14, after 16 days of treatment with DOX-loaded nanoparticles at a DOX concentration of 30 mg / kg, the tumor was markedly inhibited, and DOX also inhibited tumor proliferation, It can be seen that the side effects are very large. That is, when the concentration of DOX in the drug-loaded nanoparticles was set to 30 mg / kg, there was almost no change in the body weight of the mice. This explains that the drug-loaded nanoparticles do not cause toxic side effects to the mouse. Mouse weight is reduced by 23% at day 7, explaining that DOX has a very high side effect on mice. Similarly, when the DOX concentration was 30 mg / kg, all of the mice in this group survived for 46 days with DOX loaded nanoparticles, and all died when treated with DOX for 10 days. The mouse of the PBS group as a control group also died on the 35th day. Therefore, the drug-loaded nanoparticles can effectively inhibit tumor proliferation, have no toxic side effects on mice, and can prolong survival time of tumor mice.

Example 23 : Study of blood circulation of drug loaded target cross-linked vesicle cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k) / PEG5k-P (CDC4.9k- co- TMC19k)

Balb / C nude mouse (Laboratory Animal Center, Shanghai Institute of Bioscience) was selected for body weight of 18 ~ 20g and 4 ~ 6 weeks old. Body weight was weighed and group was evenly divided. Vesicles were constructed by different ratios of cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k) and PEG5k-P (CDC4.9k- co- TMC19k) 20%, the particle diameter is 100 nm, and the particle diameter distribution is 0.10, which indicates that the target effect is the most excellent. Target drug loading vesicles cRGD20 / CLPs, drug loading vesicle CLPs, non-transgene vesicles as control cRGD20 / PEG-PTMC and DOX.HCl were injected intravenously into the mouse via tail vein (doses of DOX 10 mg / kg ), Approximately 10 μL of blood was collected at fixed time points of 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours and the blood weight was accurately weighed through the delta rule. After centrifugation (20000 rpm, 20 minutes), the supernatant was removed, and the supernatant was removed by using a fluorescence photometer. The DOX amount at the time point was measured.

15 is the blood circulation in the mouse body of DOX-loaded target cross-linked vesicle, wherein the abscissa is the time and the ordinate is the amount of DOX (ID% / g) in the blood per g of the total DOX injection. As can be seen from FIG. 15, the circulation time of DOX.HCl was very short, so it was very difficult to detect DOX already in 2 hours, while the crosslinked vesicle was still 8 ID% / g after 24 hours. Calculations show that the elimination half-lives of target drug-loaded crosslinked vesicles, drug loaded bridged vesicles and non-glycosylated vesicles in the mouse body are 4.49, 4.26 and 1.45 hours, while DOX.HCl is only 0.27 hours And thus the target drug loading crosslinked vesicle is stable in the mouse body and has a relatively long cycle time.

Example 24: Drug loading vesicle targeting crosslinking cRGD-PEG6k-P (CDC4.6k- co -TMC18.6k) / PEG5k-P body vivo on human brain malignant glioma in mice (CDC4.9k- co -TMC19k) Distribution Study

Balb / C nude mouse (Laboratory Animal Center, Shanghai Institute of Bioscience) was selected for body weight of 18 ~ 20g and 4 ~ 6 weeks old. Body weight was weighed and group was evenly divided. 5 × 10 ⁶ U87MG human brain malignant collagen cells were subcutaneously injected and approximately 3 to 4 weeks later, when tumor size reached 100-200 mm ³ , cRGD20 / CLPs, CLPs and DOX · HCl were injected via tail vein After 4 hours, mice were sacrificed and the tumor and heart, liver, spleen, lung, and kidney were harvested, washed and weighed, and then 500 μL of 1% (w / v) Triton was added, pulverized through a homogenizer, and extracted with 900 μL of DMF (containing 20 mM of DTT and 1 M of HCl). After centrifugation (20,000 rpm, 20 minutes), the supernatant was removed, and the amount of DOX at each time point was measured with a fluorescence photometer.

16 is a biodistribution diagram of DOX-loaded target cross-linked vesicles in the human brain glioma glioma mouse. The abscissa is the tissue organs, and the ordinate is the amount of DOX (ID% / g) in the tumor or tissue per gram of total DOX injection. As shown in the figure, the DOX levels of the tumor tissues were 6.78, 2.15 and 0.82 ID% / g, respectively, after 4 hours of injection of cRGD20 / CLPs, CLPs and DOX · HCl. CRGD20 / CLPs were CLPs and DOX · HCl And 9-fold, indicating that the drug-loaded target cross-linked vesicles are relatively more accumulated in tumor sites through active targets.

Example 25 : Treatment of human brain malignant glioma mice with drug loaded target cross-linked vesicle cRGD-PEG6k-P (CDC4.6k- co- TMC18.6k) / PEG5k-P (CDC4.9k- co- Application of

Balb / C nude mouse (Laboratory Animal Center, Shanghai Institute of Bioscience) was selected for body weight of 18 ~ 20g and 4 ~ 6 weeks old. Body weight was weighed and group was evenly divided. 5 x 10 ⁶ U87MG human brain malignant glioma cells were subcutaneously injected and approximately 2 weeks later, when the size of the tumor reached 30-50 mm ³ , cRGD20 / CLPs, CLPs and non-transgenic nanobesocycles (cRGD20 / PEG - PTMC), DOX · HCl and PBS were injected intravenously into the mouse via tail vein on days 0, 4, 8 and 12, respectively (DOX drug amount 10 mg / kg). The weight of the mouse was measured every two days for 0 to 18 days, and the volume of the tumor was accurately measured through a vernier caliper. Among them, the calculation method of the tumor volume was V = (L x W x H) / 2 L is the length of the tumor, W is the width of the tumor, and H is the thickness of the tumor). Survival of mice was continuously monitored for up to 45 days.

Figure 17 is a therapeutic effect on DOX loading vesicle targeting crosslinking cRGD-PEG6k-P (CDC4.6k- co -TMC18.6k) human brain malignant glioma in mice /PEG5k-P(CDC4.9k- co -TMC19k) Wherein A is a tumor growth inhibition curve, B is a weight change curve of a mouse, and C is a mouse survival curve. As can be seen from FIG. 17, the cRGD20 / CLPs treatment group showed marked inhibition of tumor on day 18, and all of the tumors treated with CLPs or cRGD20 / PEG- b- PTMC proliferated to some extent. DOX · HCl could also inhibit tumor growth, but mice in the DOX · HCl group showed a 21% decrease in body weight at day 12, explaining that the toxic side effects on mice are very high. In contrast, mice in the cRGD20 / CLPs, CLPs, or cRGD20 / PEG- b- PTMC groups showed little change in body weight and this explains that drug loading nanoparticles have no toxic side effects on mice. The cRGD20 / CLPs treatment group survived all after 45 days, mice in the DOX · HCl group died already on the 13th day, and mice in the saline group also died on the 28th day. Thus, drug loading target bridging vesicles can effectively inhibit tumor growth, have no toxic side effects on mice, and can also prolong the survival time of tumor mice.

Example 26: Drug loading vesicle targeting crosslinking iRGD-PEG6k-P (CDC4.8k- co -TMC19.2k) / PEG5k-P treatment experiment on mice melanoma (CDC4.9k- co -TMC19k)

Vesicles consist of a mixture of iRGD-PEG6k-P (CDC4.8k- co- TMC19.2k) and PEG5k-P (CDC4.9k- co- TMC19k) at different ratios. The diameter of vesicles obtained at 0, 25%, 50% and 100% of iRGD (internalizing RGD) polymer is about 110 nm and the particle size distribution is 0.12. The iRGD molecule not only has a target orientation for tumor cells, but also mediates tumor cells and tissue penetration, so that even when a certain amount of free iRGD molecules is injected, the tumor tissue penetration of nanoparticles may also increase. The loading of DOX · HCl is obtained by the pH gradient technique, with a typical efficiency of 60-80%.

C57BL / 6 black mice (experimental animal center, Shanghai Institute of Life Science, China Academy of Sciences) were selected for body weight of 18 ~ 20g and 4-6 weeks old. After body weight was measured, groups were divided into groups of 1 × 10 ⁶ B16 melanoma cells were subcutaneously injected, and approximately one week later, when the size of the tumors reached 30-50 mm ³ , the drug loaded crosslinked vesicles containing 0, 25%, 50% and 100% of iRGD polymer, DOX HCl and PBS were injected intravenously into the melanoma mice via tail vein on days 0, 3, 6, 9 and 12 (DOX · HCl drug dose 10 mg / kg). Mice were weighed daily for 0 to 20 days and the volume of the tumor was accurately measured via a vernier caliper. The tumor volume calculation method is V = (L × W × H) / 2, where L is the length of the tumor, W is the width of the tumor, and H is the thickness of the tumor. Survival of the mice was continuously observed until day 46.

Figure 18 shows a therapeutic trial of melanoma mice of DOX loading target cell line iRGD-PEG6k-P (CDC4.8k- co- TMC19.2k) / PEG5k-P (CDC4.9k- co- TMC19k) Figure A is the tumor growth inhibition curve, Figure B is the weight change curve of the mouse, and Figure C is the mouse survival curve. As can be seen from FIG. 18, when the iRGD ratio was 50%, the iRGD50 / CLPs treatment group showed marked inhibition of tumor growth after 20 days of treatment, and when the iRGD ratio was too high or too low, It affected the intake in the tissues. Similarly, DOX · HCl also inhibited tumor proliferation, but the toxic side effects on mice were so great that the body weight of mice decreased by 20% at 8 days. All ratios of iRGD drug loaded crosslinked vesicles demonstrate that mice in each group had little or no change in body weight, and that there was no toxic side effect to the mice. The mice in the iRGD50 / CLPs group were still alive after 43 days of treatment, and the DOX · HCl group was toxic, so all mice died on the 10th day, and mice in the PBS group also died on the 29th day. Thus, the target drug loading crosslinked vesicles can effectively inhibit tumor growth, have no toxic side effects on the mice, and can also prolong the survival time of tumor mice.

Example 27 : Blood circulation of drug loaded target cross-linked vesicles cNGQ-PEG6k-P (CDC4.8k- co- TMC19.2k) / PEG5k-P (CDC4.9k- co- TMC19k) Tumor suppression experiment

And synthesis is similar to the embodiment 13 of the polymer cNGQ-PEG6k-P (CDC4.8k- co -TMC19.2k), i.e., first synthesized NHS- PEG6k-P (CDC4.8k- co -TMC19.2k ), And binds the PEG end of the polymer with a target molecule cNGQ amination reaction. As can be seen from the BCA test kit, the graft rate of cNGQ is 87%. Vesicles are constructed by mixing cNGQ-PEG6k-P (CDC4.8k- co- TMC19.2k) / PEG5k-P (CDC4.9k- co- TMC19k) at different ratios. The loading of DOX · HCl is obtained by the pH gradient technique, with a typical efficiency of 60-80%. As a result of the in vitro cell test, the target effect was the best when the cNGQ ratio was 20%, and the obtained drug loading target crosslinking vesicle (cNGQ20 / CLPs) had a half life of 4.78 hours in the circulation of nude mice . In accordance with Example 24, a lung cancer A549 tumor model was constructed subcutaneously in nude mice, and cNGQ20 / CLPs were injected through the tail vein, and cNGQ20 / CLPs were injected in 8 hours (CRGD20 / CLPs), non-target crosslinked nanoparticles (CLPs) and DOX · HCl as the control group and higher than other organs at 9 ID% / g And see the biodistribution diagram in the lung cancer mouse body of DOX loading target cross-linked vesicles of FIG.

A549 lung cancer tumor model and bioluminescent A549 lung cancer distal tumor model were constructed by subcutaneous injection in the same manner as in Example 25 and the latter could detect bioluminescence using biomedical imaging machine and further detect tumor growth status . On day 0, 4, 8, and 12, the drug was injected into the tail vein and bioluminescence detected by biomedical imaging showed that the fluorescence of the mouse lung of the cNGQ20 / CLPs treatment group was markedly weakened, indicating that cNGQ20 / CLPs Can be favorably targeted to lung cancer and effectively inhibit the proliferation of lung cancer.

Example 28 : DOX loading PEG5k-PLGA 7.8k-PCDC 1.7k gold nanorod surface modification and NIR triggered drug release

Preparation of gold nanorods modified with nanoparticles of triple block polymer PEG5k-PLGA 7.8k-PCDC 1.7k: Polymer solution (2 mL, 5 mg / mL) dissolved in DMSO was added to a gold nanodobar dispersion 5 mL, 0.1 mg / mL), stirred for 4 hours, and centrifuged twice to remove ungrafted polymer. The polymer was dispersed again in a phosphate buffer solution, and the polymer yield Respectively. The yield of the polymer modified gold rod through comparison with the sole polymer is 80% (the theory follows 100% feeding).

Drug loading of polymer modified gold nanorods: The DOX dissolved in 10%, 20%, 30% DMSO was gradually added dropwise to the obtained polymer modified gold nanorod rod solution, stirred for 30 minutes, Incubate and remove the released small molecule drug by dialysis for 12 hours. The dialysis medium is a phosphate buffer solution at a pH of 7.4, and the fluorescence detection showed that the sealing efficiency for DOX was 70-90%. FIG. 20 shows TEM images of a gold nanorod surface-modified with PEG5k-PLGA 7.8k-PCDC 1.7k, TEM , The length of the gold bar measured by the above method is about 60 nm, and it can be seen that the distribution is uniform.

DOX emission of polymer modified gold nanorods triggered by NIR: Polymer modified gold nanorods were dispersed in 10 mL of phosphate buffer solution and irradiated with infrared light with an intensity of 0.2 W / cm < ² > at 1 hour intervals and a wavelength of 808 nm for 5 min , 500 μL of the solution is centrifuged within a specific time interval, the fluorescence of the supernatant is measured, and the DOX content released is analyzed. As can be seen from the fluorescence detection, the drug release of the polymer modified gold nanorods after irradiation was 92%, much faster than the unlitrated control (the release was only 18%), It can be seen that gold nanorod materials can be applied to near-infrared-triggered drug release.

Example 29 : Application of polymer PEG 1.9k-PCDC 0.8k to a plasma resonator (SPR) sensor surface modification

The gold surface on the SPR sensor was treated with auqa regia, washed with ethanol, dried and immediately added to the THF in which the triblock copolymer PEG1.9k-PCDC0.8k (1 mL, 5 mg / mL) was dissolved Respectively. The surface of the sensor plate was cleaned three times, and the surface density of the modified PEG 1.9k on the sensor gold plate was found to be 20 nmol / cm ² Respectively. Comparing the sensor chip after the polymer modification with the conventional chip has advantages such as reduction of non-specific adsorption and improvement of stability of measurement and can be widely applied to biomedical and other fields.

Example 30 : Application of polymer P (CDC0.8k- co- CL92k) as a biodegradable support material after crosslinking

Polymer P (CDC0.8k- co- CL92k) was dissolved in trichloromethane (40 mg / mL), and a film (support material) was formed on a 1 × 1 cm ² glass plate and then dried in a vacuum dryer for 48 hours, Was completely removed and heated with a heat gun at 40 캜 for 10 minutes to cross-link the sulfur-sulfur pentavalent ring and then immersed in physiological saline for 2 weeks. As a result, the glass plate was still completely damaged, That is, the PCL film as a < / RTI > 21 is a photograph of polymer PCL and P (CDC0.8k- co- CL92k) after film formation and after immersion in physiological saline solution for 2 weeks. As a result, it can be seen that the polymer containing a sulfur-sulfur double-ring functional group in the side chain can be used as a biocompatible material because it can enhance the stability of the support material.

Claims (5)

As a carbonate polymer containing a disulfide pentagonal ring functional group in a side chain containing a cyclocarbonate monomer unit containing a disulfide pentagonal ring functional group,
The chemical structure of the carbonate polymer containing the disulfide pentagonal ring functional group in the side chain is one of the following formulas:

Wherein R1 is selected from one of the following groups:

Wherein k = 20-250 and R4 is selected from one of the following groups;

R2 is selected from one of the following groups:

,

,

,

,

;
R3 is selected from one of the following groups:

or

, Wherein a = 2, 3 or 4; b = 20-250;
The molecular weight of the carbonate polymer containing the disulfide pentagonal ring functional group in the side chain is 800 to 100000 Da,
A carbonate polymer containing a disulfide pentagonal ring functional group in its side chain. The method according to claim 1,
A carbonate polymer containing a disulfide pentagonal ring functional group on the side chain and having 4 to 50 units of cyclocarbonate monomer containing a disulfide pentagonal ring functional group on a molecular chain of a carbonate polymer containing a disulfide pentagonal ring functional group in the side chain. Wherein the carbonate polymer containing a disulfide pentagonal ring functional group in its side chain has a molecular weight of 3000 to 70000 Da, and a carbonate polymer containing a disulfide pentagonal ring functional group in the side chain as defined in claim 1 or 2. Wherein the carbonate polymer containing a disulfide pentagonal ring functional group in the side chain has a molecular weight of 5,000 to 100,000 Da, and a carbonate polymer containing a disulfide pentagonal ring functional group in the side chain according to any one of claims 1 to 4. Wherein the carbonate polymer containing a disulfide pentagonal ring functional group in its side chain has a molecular weight of 800 to 10,000 Da. 2. The composition for coating a biochip as claimed in claim 1 or 2, wherein the carbonate polymer contains a disulfide pentagonal ring functional group.

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